Biodentine Induces Human Dental Pulp Stem Cell Differentiation through Mitogen-activated Protein Kinase and Calcium-/Calmodulin-dependent Protein Kinase II Pathways Zhirong Luo, MS,* Meetu R. Kohli, BDS, DMD, † Qing Yu, DDS, PhD,* Syngcuk Kim, DDS, PhD, † Tiejun Qu, DDS, PhD,* and Wen-xi He, DDS, PhD* Abstract Introduction: Biodentine (Septodont, Saint-Maur-des- Foss es, France), a new tricalcium silicate cement formu- lation, has been introduced as a bioactive dentine substitute to be used in direct contact with pulp tissue. The aim of this study was to investigate the response of human dental pulp stem cells (hDPSCs) to the material and whether mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-kB), and calcium-/ calmodulin-dependent protein kinase II (CaMKII) signal pathways played a regulatory role in Biodentine-induced odontoblast differentiation. Methods: hDPCs obtained from impacted third molars were incubated with Bio- dentine. Odontoblastic differentiation was evaluated by alkaline phosphatase activity, alizarin red staining, and quantitative real-time reverse-transcriptase poly- merase chain reaction for the analysis of messenger RNA expression of the following differentiation gene markers: osteocalcin (OCN), dentin sialophosprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialo- protein (BSP). Cell cultures in the presence of Biodentine were exposed to specific inhibitors of MAPK (U0126, SB203580, and SP600125), NF-kB (pyrrolidine dithiocar- bamate), and CaMKII (KN-93) pathways to evaluate the regulatory effect on the expression of these markers and mineralization assay. Results: Biodentine significantly increased alkaline phosphatase activity and mineralized nodule formation and the expression of OCN, DSPP, DMP1, and BSP. The MAPK inhibitor for extracellular signal-regulated kinase 1/2 (U0126) and Jun N-terminal ki- nase (SP600125) significantly decreased the Biodentine- induced mineralized differentiation of hDPSCs and OCN, DSPP, DMP1, and BSP messenger RNA expression, whereas p38 MAPK inhibitors (SB203580) had no effect. The CaMKII inhibitor KN-93 significantly attenuated and the NF-kB inhibitor pyrrolidine dithiocarbamate further enhanced the up-regulation of Biodentine-induced gene expression and mineralization. Conclusions: Biodentine is a bioactive and biocompatible material capable of inducing odontoblast differentiation of hDPSCs. Our results indicate that this induction is regulated via MAPK and CaMKII pathways. (J Endod 2014;40:937–942) Key Words Biodentine, biosilicate cement, calcium-/calmodulin-dependent protein kinase II, pathway, human dental pulp stem cells, mitogen-activated protein kinase pathway, nuclear factor-kappa B pathway T he biocompatibility of a material is an important property when placed in direct con- tact with the pulp. In the last decade, mineral trioxide aggregate (MTA) has become the material of choice for this purpose. It has shown superior prognostic results when used clinically in direct pulp capping procedures (1, 2). In vivo and in vitro studies have shown the formation of a dentin bridge under exposed sites when sealed with MTA (2–4). This ability is attributed to its sealability and biocompatibility because of the presence of tricalcium silicate (5). However, the major drawbacks of MTA are its handling properties, long setting time, and discoloration of the remaining tooth struc- ture. In recent years, bioactive tricalcium silicate cements have been introduced that claim to overcome these limitations. One such material is Biodentine (BD; Septodont, Saint-Maur-des-Foss  es, France). It is marketed as a bioactive dentin substitute with active biosilicate technology. It is a tricalcium silicate cement that the manufacturer claims promotes pulp healing and remineralization by the production of reactionary dentin and dentin bridges. In a recent study, Nowicka et al (6) showed capped iatro- genic pulp exposure in human molars scheduled for extraction with MTA and BD. The histological analysis of these teeth showed completed dentin bridge formation and a lack of inflammatory cells. MTA and BD had a similar efficacy in this experimental model. The material is dispensed in a powder liquid form in a single-dose capsule to be triturated in an amalgamator for 30 seconds. The powder is mainly composed of tricalcium silicate, calcium carbonate, and zirconium oxide. The liquid contains water, calcium chloride (used as a setting accelerator), and a modified polycarbox- ylate (a superplasticizing agent) (7). The total handling time (ie, mixing, placing, and setting) is 12 minutes as described by the manufacturer. Consequently, BD has 2 main benefits over other products: its reduced setting time (a few minutes From the *Department of Operative Dentistry and Endodontics, Fourth Military Medical University, Xian, China; and † Department of Endodontics, University of Pennsylvania, Philadelphia, Pennsylvania. Address requests for reprints to Dr Wen-xi He, Department of Operative Dentistry and Endodontics, Fourth Military Medical University, 145 Chang-le Xi Road, Xian 710032, PR China. E-mail address: hewenxi@fmmu.edu.cn 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.11.022 Basic Research—Biology JOE — Volume 40, Number 7, July 2014 Biodentine and hDPSC Differentiation 937